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Oct 24, 2011
Mating type switching in S.pombe
PMID: 3561486
There are two mating types in S.pombe: M(minus) and P(plus).
The P and M mating types are controlled by the mat1-P mat1-M alleles of the mat1 mating-type
locus, which is a part of mat1-mat2-mat3 cluster on chromosome II.
The mating-type genes of S. pombe are found at three locations in the same chromosomal region.
These genes are in an active configuration at the mat1 locus and in an inactive configuration at
the mat2 and mat3 loci.
The mechanism that represses transcription of mat2 and mat3 also inactivates other promoters
introduced nearby and is accompanied by a block to meiotic recombination in the mat2 mat3
interval. Also there is a mechanism that prevents homologous recombination in the region during
meiosis.
The mat1 and mat2/mat3 loci are genetically separated by 1cM (in reality they are 3cM apart),
but mat2 and mat3 loci behave genetically as a single locus (even though in reality they are 3cM
apart).
Wild-type strains interchange mat1 alleles every other generation using mat2 and mat3 as donors
of genetic information. Only one grandchild is switched of the four progeny.
Mating type switching is triggered by a
double-stranded break (DSB) within H1 of
mat1 locus.
Mechanism:
 In Pu --grandmother
One of the two chains of DNA molecule
(lagging strand) upon its replication acquires
UU. There is a pause site upstream of H1 that is requited for pausing of DNA replication at the
replication fork. This pausing is needed for the addition of UU. This UU chromosomal marking
on a single DNA strand is called imprinting.
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 This imprinted DNA is inherited by Ps and WT DNA is inherited by Pu cell
 In Ps:
This imprinted DNA is replicated as a template for the leading strand. DNApol is
stalled upon encountering UU and a double-stranded break is introduced in H1 of mat1. This
double-stranded break is repaired via gene
conversion. Where the 3’ end of the broken
strand is resected and then the resulting
single-stranded DNA invades H1 region of
mat2P or mat3M.
Lab strains mat2Δ mat3Δ still have dsDNA
break, but it is repaired probably by a nonhomologous end-joining (NHEJ), since the
donor cassettes are missing.
Question: What would happen if
homologous recombination instead of gene
conversion is allowed to happen at the
“homology regions”?
Answer: It would produce deletions to the mat
locus and inability to switch to the opposite
mating type. Since there are essential genes
between mat1 and mat2, such a mutant would
die (so one could look for conditional
mutants).
Diagram on the board
The interval between mat2 and mat3 is called
K-region. K-region is highly homologous to
centromeric region and we know that
homologous recombination at centromeres is suppressed.
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Problem on the board:
How would you identify the importance of K-region in:
a) silencing
b) mating-type switching
Silencing
 Design URA4 with homology arms that would delete most of the K-region
PMID:9258669
 Transform ura4Δ leu1Δ clr4∆ strain that is a h90 (switching strain). Why use a silencing
defective strain? Because not knowing whether or not the inserted ura4+ gene will be
silencing, this is the prudent thing to do.
 Perform PCR to verify if the insert is in the correct place
 Select on -Ura, -Leu
 Then cross to a clr4+ ura4∆ leu1∆ to get wild-type clr4+
 Plate on YPD (to look at the total) and then replica-plate to 5-FOA, –Ura, both.
Results: 30% of colonies grow on 5FOA and 70% on –Ura. None grow on both.
Conclusion: The K-region is important for stable silencing and that in its absence, cells take on
one of two heritable states. Assuming the DNA sequence is identical of the two states, this is an
example of an epigenetic switch.
Mating-type switching
Now you want to figure out whether K-region’s silencing or non-silencing is correlated with the
ability to switch to the opposite mating type
 Mate cells from “silenced” and “non-silenced” colonies on plates with M and P strains
(these are the lab strain—stable mat 1-P(M) mat2-3Δ::LEU1)
 Mating is performed on nitrogen-poor medium necessary for the induction of mating and
sporulation
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 Use iodine-vapor staining to look for sporulating cells.
Question: How will you account for “non-silenced” haploids that sporulate without mating and
are iodine-stained? Derepression of silenced M and P genes cause haploid cells to behave like
mated diploid cells and trigger meiosis and sporulation.
Answer: Select diploids on –Ura –Leu medium (nitrogen-poor) and in the presence of iodinevapors.
Question: How will you determine if both mat2 and mat3 are utilized equally in “nonsilencers”?
Answer: When you mate them to P or M laboratory strains and select for diploids you can look
whether mating to P or M preferentially produces spores. You need to have a control: WT mated
to P and M strains.
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